Hypertension represents a major public health concern. To date, each form of inherited hypertension has been linked to defective salt handling in the kidney. This application seeks to explore the mechanisms involved in the regulation of one of the key salt-handling transporters in the kidney, the sodium chloride co- transporter (NCC) located in distal convoluted tubule. Previous studies have reported decreased NCC activity in response to the presence of WNK4 (a kinase associated with a form of genetic hypertension called Gordon's Syndrome) and phorbol esters (chemical analogs of diacylglycerol, a common pathway for hormonal stimulation) but the mechanism by which NCC activity is reduced is unclear. This application will seek to demonstrate that WNK4 and phorbol esters decrease NCC activity by decreasing the amount of NCC expressed at the cell surface, a process that occurs by enhancing the ubiquitination of NCC, thereby promoting degradation of the co-transporter. The experimental model will be a cell model utilizing an immortalized mouse distal convoluted tubule cell line with native NCC activity and intact regulatory pathways. The association between activity, surface expression, and ubiquitination will be established by measuring NCC activity via radiotracer uptake, NCC cell surface expression via biotinylation and immunofluorescence, and ubiquitination via immunoprecipitation and immunoblotting in cells gene-silenced for WNK4 or treated with phorbol esters. The mechanisms of NCC ubiquitination will also be determined, including sites of ubiquitination and required enzymes. This project will help train Dr. Benjamin Ko as a physician-scientist and facilitate his transition to an independent investigator and contributing member of the scientific community. Public Health Relevance Hypertension represents a tremendous public health concern with staggering morbidity and mortality. As inherited hypertension is intimately tied to sodium reabsorption and many of our treatments for hypertension inhibit NCC activity, understanding the regulation of NCC activity will give us insight into the pathogenesis of hypertension and may one day provide further treatment avenues to control hypertension.